In the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D), an insulin peptide (B:9-23) is a major target for pathogenic CD4 + T cells. However, there is no consensus on the relative importance of the various positions or "registers" this peptide can take when bound in the groove of the NOD MHCII molecule, IA g7 . This has hindered structural studies and the tracking of the relevant T cells in vivo with fluorescent peptide-MHCII tetramers. Using mutated B:9-23 peptides and methods for trapping the peptide in particular registers, we show that most, if not all, NOD CD4 + T cells react to B:9-23 bound in low-affinity register 3. However, these T cells can be divided into two types depending on whether their response is improved or inhibited by substituting a glycine for the B:21 glutamic acid at the p8 position of the peptide. On the basis of these findings, we constructed a set of fluorescent insulin-IA g7 tetramers that bind to most insulin-specific Tcell clones tested. A mixture of these tetramers detected a high frequency of B:9-23-reactive CD4 + T cells in the pancreases of prediabetic NOD mice. Our data are consistent with the idea that, within the pancreas, unique processing of insulin generates truncated peptides that lack or contain the B:21 glutamic acid. In the thymus, the absence of this type of processing combined with the low affinity of B:9-23 binding to IA g7 in register 3 may explain the escape of insulin-specific CD4 + T cells from the mechanisms that usually eliminate self-reactive T cells.antigen processing | autoimmunity | T cell receptor | self tolerance I n human type 1 diabetes (T1D) and in the nonobese diabetic (NOD) mouse model of the disease, insulin is a major autoantigen for both B cells and T cells (reviewed in refs. 1, 2). A peptide from the insulin beta chain (B:9-23) has been known for many years to be the major target of insulin-reactive CD4 + T cells in NOD T1D . However, the data suggest that this peptide can bind to the NOD class II major histocompatibility (MHCII), IA g7 , in multiple positions or "registers" within the peptide binding groove (3-7). These registers are defined by the peptide amino acids occupying positions p1-p9 in the groove, which include the "anchor" amino acids at p1, p4, p6, and p9, whose side chains interact with compatible pockets in the MHC groove (8, 9). For an individual peptide, each shift in register puts a new set of peptide amino acids into these anchor positions and brings a different set of peptide amino acid side chains to the surface for potential T-cell recognition, generating a unique ligand. Defining which of the possible B:9-23 binding register(s) in the IA g7 groove create the ligand(s) for diabetogenic insulin-reactive T cells has been difficult, leading to uncertainty in exactly how this peptide is processed and presented to T cells in the pancreas and the inability to construct the relevant fluorescent insulin-IA g7 multimers for in vivo tracking the autoimmune B:9-23-specific T cells.Recently, using techniques to trap versio...

Allergic airway inflammation and hyperreactivity are modulated by γδ T cells, but different experimental parameters can influence the effects observed. For example, in sensitized C57BL/6 and BALB/c mice, transient depletion of all TCR-δ+ cells just before airway challenge resulted in airway hyperresponsiveness (AHR), but caused hyporesponsiveness when initiated before i.p. sensitization. Vγ4+ γδ T cells strongly suppressed AHR; their depletion relieved suppression when initiated before challenge, but not before sensitization, and they suppressed AHR when transferred before challenge into sensitized TCR-Vγ4−/−/6−/− mice. In contrast, Vγ1+ γδ T cells enhanced AHR and airway inflammation. In normal mice (C57BL/6 and BALB/c), enhancement of AHR was abrogated only when these cells were depleted before sensitization, but not before challenge, and with regard to airway inflammation, this effect was limited to C57BL/6 mice. However, Vγ1+ γδ T cells enhanced AHR when transferred before challenge into sensitized B6.TCR-δ−/− mice. In this study Vγ1+ cells also increased levels of Th2 cytokines in the airways and, to a lesser extent, lung eosinophil numbers. Thus, Vγ4+ cells suppress AHR, and Vγ1+ cells enhance AHR and airway inflammation under defined experimental conditions. These findings show how γδ T cells can be both inhibitors and enhancers of AHR and airway inflammation, and they provide further support for the hypothesis that TCR expression and function cosegregate in γδ T cells.

The gammadelta T-cell receptors (TCRs) are limited in their diversity, suggesting that their natural ligands may be few in number. Ligands for gammadeltaTCRs that have thus far been determined are predominantly of host rather than foreign origin. Correlations have been noted between the Vgamma and/or Vdelta genes a gammadelta T cell expresses and its functional role. The reason for these correlations is not yet known, but several different mechanisms are conceivable. One possibility is that interactions between particular TCR-V domains and ligands determine function or functional development. However, a recent study showed that at least for one ligand, receptor specificity is determined by the complementarity-determining region 3 (CDR3) component of the TCR-delta chain, regardless of the Vgamma and/or Vdelta. To determine what is required in the TCR for other specificities and to test whether recognition of certain ligands is connected to cell function, more gammadeltaTCR ligands must be defined. The use of recombinant soluble versions of gammadeltaTCRs appears to be a promising approach to finding new ligands, and recent results using this method are reviewed.

The Vγ4+ pulmonary subset of γδ T cells regulates innate airway responsiveness in the absence of αβ T cells. We now have examined the same subset in a model of allergic airway disease, OVA-sensitized and challenged mice that exhibit Th2 responses, pulmonary inflammation, and airway hyperreactivity (AHR). In sensitized mice, Vγ4+ cells preferentially increased in number following airway challenge. Depletion of Vγ4+ cells before the challenge substantially increased AHR in these mice, but had no effect on airway responsiveness in normal, nonchallenged mice. Depletion of Vγ1+ cells had no effect on AHR, and depletion of all TCR-δ+ cells was no more effective than depletion of Vγ4+ cells alone. Adoptively transferred pulmonary lymphocytes containing Vγ4+ cells inhibited AHR, but lost this ability when Vγ4+ cells were depleted, indicating that these cells actively suppress AHR. Eosinophilic infiltration of the lung and airways, or goblet cell hyperplasia, was not affected by depletion of Vγ4+ cells, although cytokine-producing αβ T cells in the lung increased. These findings establish Vγ4+ γδ T cells as negative regulators of AHR and show that their regulatory effect bypasses much of the allergic inflammatory response coincident with AHR.

It has been reported that the IgE response to allergens is influenced by γδ T cells. Intrigued by a study showing that airway challenge of mice with OVA induces in the spleen the development of γδ T cells that suppress the primary IgE response to i.p.-injected OVA-alum, we investigated the γδ T cells involved. We found that the induced IgE suppressors are contained within the Vγ4+ subset of γδ T cells of the spleen, that they express Vδ5 and CD8, and that they depend on IFN-γ for their function. However, we also found that normal nonchallenged mice harbor IgE-enhancing γδ T cells, which are contained within the larger Vγ1+ subset of the spleen. In cell transfer experiments, airway challenge of the donors was required to induce the IgE suppressors among the Vγ4+ cells. Moreover, this challenge simultaneously turned off the IgE enhancers among the Vγ1+ cells. Thus, airway allergen challenge differentially affects two distinct subsets of γδ T cells with nonoverlapping functional potentials, and the outcome is IgE suppression.

Pulmonary gammadelta T cells protect the lung and its functions, but little is known about their distribution in this organ and their relationship to other pulmonary cells. We now show that gammadelta and alphabeta T cells are distributed differently in the normal mouse lung. The gammadelta T cells have a bias for nonalveolar locations, with the exception of the airway mucosa. Subsets of gammadelta T cells exhibit further variation in their tissue localization. gammadelta and alphabeta T cells frequently contact other leukocytes, but they favor different cell-types. The gammadelta T cells show an intrinsic preference for F4/80+ and major histocompatibility complex class II+ leukocytes. Leukocytes expressing these markers include macrophages and dendritic cells, known to function as sentinels of airways and lung tissues. The continuous interaction of gammadelta T cells with these sentinels likely is related to their protective role.

In type 1 diabetes (T1D), proinsulin is a major autoantigen and the insulin B:9–23 peptide contains epitopes for CD4 T cells in both mice and humans. This peptide requires C-terminal mutations for uniform binding in the proper position within the mouse IAg7 or human DQ8 MHCII peptide grooves and for strong CD4 T cell stimulation. Here we present structures showing how these mutations control CD4 T cell receptor (TCR) binding to these MHCII-peptide complexes. Our data reveal striking similarities between mouse and human CD4 TCRs in their interactions with these ligands. We also show how fusions between fragments of B:9–23 and of proinsulin C-peptide create chimeric peptides with activities as strong or stronger than the mutated insulin peptides. We propose transpeptidation in the lysosome as a mechanism that could accomplish these fusions in vivo, similar to the creation fused peptide epitopes for MHCI presentation shown to occur by transpeptidation in the proteasome. Were this mechanism unique to the pancreas and absent in the thymus, it could provide an explanation for how diabetogenic T cells escape negative selection during development but find their modified target antigens in the pancreas to cause T1D.

Mice sensitized and challenged with OVA were used to investigate the role of innate T cells in the development of allergic airway hyperresponsiveness (AHR). AHR, but not eosinophilic airway inflammation, was induced in T cell-deficient mice by small numbers of cotransferred γδ T cells and invariant NKT cells, whereas either cell type alone was not effective. Only Vγ1+Vδ5+ γδ T cells enhanced AHR. Surprisingly, OVA-specific αβ T cells were not required, revealing a pathway of AHR development mediated entirely by innate T cells. The data suggest that lymphocytic synergism, which is key to the Ag-specific adaptive immune response, is also intrinsic to T cell-dependent innate responses.